scholarly journals Fluorescence studies on the role of tryptophan in heterogeneous nuclear ribonucleoprotein particles of HeLa cells

1989 ◽  
Vol 263 (1) ◽  
pp. 279-283 ◽  
Author(s):  
J Schenkel ◽  
I Appel ◽  
R Schwarzwald ◽  
E K F Bautz ◽  
J Wolfrum ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Hela Cells ◽  
Rna Binding ◽  
Tryptophan Fluorescence ◽  
Fluorescence Decay ◽  
Fluorescence Studies ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
Tryptophan Residues ◽  
Nuclear Ribonucleoprotein

The 40 S heterogeneous nuclear ribonucleoprotein (hnRNP) particles from HeLa cells reveal tryptophan fluorescence with a bi-exponential decay, indicating that only a few of the ‘core’ proteins contain tryptophan residues. The presence of tryptophan residues distinguishes hnRNP particles from nucleosomes, with which they otherwise share a number of properties. This difference, however, is not essential for protein-RNA binding, as the fluorescence decay remains unchanged when hnRNP particles are dissociated into protein and RNA. However, the Stern-Volmer quenching constant is doubled upon salt dissociation, i.e. tryptophan residues become more accessible to solvent. Thus tryptophan quenching is a useful parameter for monitoring protein-protein interactions in hnRNP particles.

scholarly journals Heterogeneous Nuclear Ribonucleoprotein M Facilitates Enterovirus Infection

2015 ◽  
Vol 89 (14) ◽  
pp. 7064-7078 ◽  
Author(s):  
Julienne M. Jagdeo ◽  
Antoine Dufour ◽  
Gabriel Fung ◽  
Honglin Luo ◽  
Oded Kleifeld ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Hela Cells ◽  
Rna Binding ◽  
Rna Stability ◽  
General Strategy ◽  
Entry Site ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Novel Target ◽  
Nuclear Ribonucleoprotein

ABSTRACTPicornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host antiviral defenses. Here, using a proteomics-based approach known as TAILS to identify protease-generated neo-N-terminal peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoprotein M (hnRNP M), a nucleocytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNP M is cleavedin vitroby poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in the hearts of CVB3-infected mice. hnRNP M relocalizes from the nucleus to the cytoplasm during poliovirus infection. Finally, depletion of hnRNP M using small interfering RNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus internal ribosome entry site translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNP M is a general strategy utilized by picornaviruses to facilitate viral infection.IMPORTANCEEnteroviruses, a member of the picornavirus family, are RNA viruses that cause a range of diseases, including respiratory ailments, dilated cardiomyopathy, and paralysis. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. Here, we identify hnRNP M as a novel target of a viral proteinase. We demonstrate that the virus subverts the function of hnRNP M and redirects it to a step in the viral life cycle. We propose that cleavage of hnRNP M is a general strategy that picornaviruses use to facilitate infection.

scholarly journals The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles.

1994 ◽  
Vol 14 (1) ◽  
pp. 518-533 ◽  
Author(s):  
M Huang ◽  
J E Rech ◽  
S J Northington ◽  
P F Flicker ◽  
A Mayeda ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Studies ◽  
C Protein ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
C Proteins ◽  
Assembly Intermediate ◽  
Nuclear Ribonucleoprotein

A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site size of 230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt) into a unique 19S triangular complex. In the absence of this unique structure, the basic A- and B-group proteins bind RNA to form several different artifactual structures which are not present in preparations of native hnRNP and which do not function in hnRNP assembly. Three (A2)3B1 tetramers bind the 19S complex to form a 35S assembly intermediate. Following UV irradiation to immobilize the C proteins on the packaged RNA, the 19S triangular complex is recovered as a remnant structure from both native and reconstituted hnRNP particles. C protein-RNA complexes composed of three, six, or nine tetramers (one, two, or three triangular complexes) nucleate the stoichiometric assembly of monomer, dimer, and trimer hnRNP particles. The binding of C-protein tetramers to RNAs longer than 230 nt is through a self-cooperative combinatorial mode. RNA packaged in the 19S complex and in 40S hnRNP particles is efficiently spliced in vitro. These findings demonstrate that formation of the triangular C protein-RNA complex is an obligate first event in the in vitro and probably the in vivo assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts.

scholarly journals Recognition of subsets of the mammalian A/B-type core heterogeneous nuclear ribonucleoprotein polypeptides by novel autoantibodies

1996 ◽  
Vol 320 (3) ◽  
pp. 761-767 ◽  
Author(s):  
Amalia DANGLI ◽  
Angeliki PLOMARITOGLOU ◽  
Efrosini BOUTOU ◽  
Neratzoula VASSILIADOU ◽  
Haralampos M. MOUTSOPOULOS ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Peptide Mapping ◽  
Protein A ◽  
Major Protein ◽  
Protein Species ◽  
Two Dimensional Gel Electrophoresis ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
Nuclear Ribonucleoprotein

The structurally related A/B-type core heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptides of 34–39 kDa (A1, A2, B1 and B2) belong to a family of RNA-binding proteins that are major components of 40 S hnRNP complexes. By two-dimensional gel electrophoresis and peptide mapping analysis we compared each member of the A/B-type core proteins in the human and rat liver cells. This comparison revealed the unique presence in rat cells of major protein species, referred to as mBx polypeptides, that appeared as three charge isoforms at a position corresponding to the minor HeLa B1b protein spot. In addition, clear differences in the ratios of the A1 polypeptide to the A1b isoform were observed. The detection, in sera of patients with rheumatic autoimmune diseases, of two novel autoantibody specificities, one recognizing solely B2 protein and the second both the B2 and mBx polypeptides, helped to identify mBx proteins as new A/B-type hnRNP components, immunologically related to B2 protein. A common immunoreactive V8 protease peptide of approx. 17 kDa has been identified in B2 and mBx hnRNP polypeptides. mBx protein species are identified in cells of murine origin, and have a ubiquitous tissue distribution and developmental appearance.

scholarly journals The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles

1994 ◽  
Vol 14 (1) ◽  
pp. 518-533
Author(s):  
M Huang ◽  
J E Rech ◽  
S J Northington ◽  
P F Flicker ◽  
A Mayeda ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Studies ◽  
C Protein ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
C Proteins ◽  
Assembly Intermediate ◽  
Nuclear Ribonucleoprotein

A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site size of 230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt) into a unique 19S triangular complex. In the absence of this unique structure, the basic A- and B-group proteins bind RNA to form several different artifactual structures which are not present in preparations of native hnRNP and which do not function in hnRNP assembly. Three (A2)3B1 tetramers bind the 19S complex to form a 35S assembly intermediate. Following UV irradiation to immobilize the C proteins on the packaged RNA, the 19S triangular complex is recovered as a remnant structure from both native and reconstituted hnRNP particles. C protein-RNA complexes composed of three, six, or nine tetramers (one, two, or three triangular complexes) nucleate the stoichiometric assembly of monomer, dimer, and trimer hnRNP particles. The binding of C-protein tetramers to RNAs longer than 230 nt is through a self-cooperative combinatorial mode. RNA packaged in the 19S complex and in 40S hnRNP particles is efficiently spliced in vitro. These findings demonstrate that formation of the triangular C protein-RNA complex is an obligate first event in the in vitro and probably the in vivo assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts.

scholarly journals Heterogeneous Nuclear Ribonucleoprotein (hnRNP) E1 Binds to hnRNP A2 and Inhibits Translation of A2 Response Element mRNAs

2006 ◽  
Vol 17 (8) ◽  
pp. 3521-3533 ◽  
Author(s):  
Linda D. Kosturko ◽  
Michael J. Maggipinto ◽  
George Korza ◽  
Joo Won Lee ◽  
John H. Carson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
In Vitro Translation ◽  
Dependent Manner ◽  
Response Element ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Hnrnp E1

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.

scholarly journals Heterogeneous nuclear ribonucleoprotein D0B is a sequence-specificDNA-binding protein

1999 ◽  
Vol 338 (2) ◽  
pp. 417-425 ◽  
Author(s):  
Mate TOLNAY ◽  
Lyudmila A. VERESHCHAGINA ◽  
George C. TSOKOS
Keyword(s):  
Dna Sequences ◽  
Rna Binding ◽  
Binding Protein ◽  
Double Helix ◽  
Physiological Role ◽  
Single Stranded Dna ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Double Stranded Dna ◽  
Nuclear Ribonucleoprotein

Complement receptor 2 (CR2) is important in the regulation of the B lymphocyte response; the regulation of its expression is therefore of central importance. We recently reported that a 42 kDa heterogeneous nuclear ribonucleoprotein (hnRNP) is involved in the transcriptional regulation of the human CR2 gene [Tolnay, Lambris and Tsokos (1997) J. Immunol. 159, 5492–5501]. We cloned the cDNA encoding this protein and found it to be identical with hnRNP D0B, a sequence-specific RNA-binding protein. By using a set of mutated oligonucleotides, we demonstrated that the recombinant hnRNP D0B displays sequence specificity for double-stranded oligonucleotide defined by the CR2 promoter. We conducted electrophoretic mobility-shift assays to estimate the apparent Kd of hnRNP D0B for the double-stranded DNA motif and found it to be 59 nM. Interestingly, hnRNP D0B displayed affinities of 28 and 18 nM for the sense and anti-sense strands of the CR2 promoter-defined oligonucleotide respectively. The significantly greater binding affinity of hnRNP D0B for single-stranded DNA than for double-stranded DNA suggests that the protein might melt the double helix. The intranuclear concentration of sequence-specific protein was estimated to be 250–400 nM, indicating that the protein binds to the CR2 promoter in vivo. Co-precipitation of a complex formed in vivo between hnRNP D0B and the TATA-binding protein demonstrates that hnRNP D0B interacts with the basal transcription apparatus. Our results suggest a new physiological role for hnRNP D0B that involves binding to double- and single-stranded DNA sequences in a specific manner and functioning as a transcription factor.

The Steady-State and Decay Characteristics of Protein Tryptophan Fluorescence from Algae

1988 ◽  
Vol 42 (8) ◽  
pp. 1405-1412 ◽  
Author(s):  
M. Baek ◽  
W. H. Nelson ◽  
P. E. Hargraves ◽  
J. F. Tanguay ◽  
S. L. Suib
Keyword(s):  
Steady State ◽  
Tryptophan Fluorescence ◽  
Fluorescence Decay ◽  
Direct Application ◽  
Fluorescence Lifetimes ◽  
Bacterial Protein ◽  
Steady State Fluorescence ◽  
Tryptophan Residues ◽  
Rapid Characterization

The intrinsic steady-state fluorescence due to tryptophan has been obtained from monospecific cultures of fourteen plankton algae of various genera. Fluorescence decay profiles of protein tryptophan residues were obtained for eight marine plankton algae. Each organism exhibits a strong maximum in its emission spectrum at 320–340 nm when excited at 290 nm. Iodide quenching and denaturization experiments with 8 M urea provide strong evidence for the assignment of the 320–340 nm fluorescence to protein tryptophan. Most importantly, the decay of this bacterial protein tryptophan fluorescence has been described. The observation that characteristic protein-tryptophan fluorescence lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of algae. Direct application will likely be found in combination with the measurement of other luminescence parameters.

Both RNA-Binding Domains in Heterogeneous Nuclear Ribonucleoprotein A1 Contribute Toward Single-Stranded-RNA Binding

Biochemistry ◽  
1994 ◽  
Vol 33 (27) ◽  
pp. 8272-8281 ◽  
Author(s):  
Yousif Shamoo ◽  
Norzehan Abdul-Manan ◽  
Ann M. Patten ◽  
Janet K. Crawford ◽  
Matthew C. Pellegrini ◽  
...  
Keyword(s):  
Rna Binding ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Binding Domains ◽  
Rna Binding Domains ◽  
Nuclear Ribonucleoprotein
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